Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane Martin Knöpfel a , Joanna P. Davies b , Phu T. Duong c , Lisbet Kværnø a , Erick M. Carreira a , Michael C. Phillips c , Yiannis A. Ioannou b , Helmut Hauser d, ⁎ a Laboratorium für Organische Chemie, ETH Zürich, CH-8093 Zürich, Switzerland b Department of Human Genetics, The Mount Sinai School of Medicine, New York, NY 10029, USA c Children’s Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318, USA d Lipideon Biotechnology AG, Fluehgasse 63, 8008 Zürich, Switzerland Received 26 January 2007; received in revised form 1 May 2007; accepted 2 May 2007 Available online 23 June 2007 Abstract We compared cholesterol uptake into brush border membrane vesicles (BBMV) made from the small intestines of either wild-type or Niemann–Pick C1-like 1 (NPC1L1) knockout mice to elucidate the contribution of NPC1L1 to facilitated uptake; this uptake involves cholesterol transport from lipid donor particles into the BBM of enterocytes. The lack of NPC1L1 in the BBM of the knockout mice had no effect on the rate of cholesterol uptake. It follows that NPC1L1 cannot be the putative high-affinity, ezetimibe-sensitive cholesterol transporter in the brush border membrane (BBM) as has been proposed by others. The following findings substantiate this conclusion: (I) NPC1L1 is not a brush border membrane protein but very likely localized to intracellular membranes; (II) the cholesterol absorption inhibitor ezetimibe and its analogues reduce cholesterol uptake to the same extent in wild-type and NPC1L1 knockout mouse BBMV. These findings indicate that the prevailing belief that NPC1L1 facilitates intestinal cholesterol uptake into the BBM and its interaction with ezetimibe is responsible for the inhibition of this process can no longer be sustained. © 2007 Elsevier B.V. All rights reserved. Keywords: Intestinal cholesterol absorption; Niemann–Pick C1 like 1; wt and NPC1L1 −/− mouse brush border membrane vesicle; Inhibitors of cholesterol absorption; Ezetimibe; Apolipoprotein A-I 1. Introduction There is a conspicuous gap in our understanding of cholesterol homeostasis: the pathway of cholesterol absorption in the small intestine is still poorly understood. The uptake 1 of cholesterol as well as other water-insoluble dietary lipids from the lumen of the small intestine into the brush border membrane (BBM) of intestinal epithelial cells (enterocytes) is generally accepted as a protein-mediated process [1] albeit, important mechanistic details of this process have remained elusive. In particular, the identity of the proteins involved in intestinal Biochimica et Biophysica Acta 1771 (2007) 1140 – 1147 www.elsevier.com/locate/bbalip Abbreviations: apo A-I, apolipoprotein A-I; BBM, brush border membrane; BBMV, brush border membrane vesicle(s); CD36, cluster determinant 36; COE, cholesteryl oleyl ether; DMSO, dimethyl sulfoxide; DTT, 1,4-dithio-rac-threitol; EGTA, ethylene glycol bis (β-aminomethyl ether)-N,N,N′,N′-tetraacetic acid; HDL, high-density lipoprotein; mBBMV, mouse brush border membrane vesicle(s); NPC1L1, Niemann–Pick C1 like 1; PC, phosphatidylcholine; SDS, sodium dodecyl sulfate; SR-BI, scavenger receptor class B, type I; SUV, small unilamellar vesicle(s); TCA, trichloro acetic acid; TME, Tris buffer containing mannitol and EGTA; TTBS, Tween 20 in Tris-buffered saline; wt, wild-type ⁎ Corresponding author. Tel.: +41 44 9263778; fax: +41 44 9263993. E-mail address: helmut.hauser@lipideon.com (H. Hauser). 1 Usually the terms lipid uptake and lipid absorption are used interchange- ably. Here the term cholesterol uptake is defined as the process of cholesterol transfer from the donor particle to the BBM. In contrast, cholesterol absorption is a multiple transport process of cholesterol from the lumen of the small intestine to the lymph and circulatory system. By this definition cholesterol uptake is the first reaction in the cascade of transport processes comprising cholesterol absorption. Cholesterol uptake is measured in vitro whereas cholesterol absorption is measured in vivo. 1388-1981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2007.05.011